Medicament containing a tissue inhibitor of metalloproteinases-2 (timp-2) as an osteoanabolically active substance

ABSTRACT

The invention relates to TIMP-2) as an osteoanabolically active peptide for use as a medicament for treating bone defects, bone diseases and for improving bone regeneration.

[0001] The invention relates to a medicament and a diagnostic agentcontaining tissue inhibitor of metalloproteinases-2 (TIMP-2) as anosteoanabolically active peptide preparation, and the use thereof.

[0002] TIMP-2 belongs to the family of “tissue inhibitors ofmetalloproteinases”, of which four different peptides (TIMP-1 to TIMP-4)having different biological functions have been known to date.

[0003] The primary structure of TIMP-2 could be elucidated for variousorganisms, such as humans (Liotta et al., 1991), rats (Roswit et al.,1992), and mice (Kishi et al., 1991). The peptide contains a total of 12cysteines which are linked to one another through 6 cystine bridges.

[0004] The biological functions of TIMP-2 include, inter alia, theinhibition of active matrix metalloproteinases (MMPs). The term MMPsrefers to a group of zinc-dependent endoproteinases which are capable ofdegrading the extracellular matrix. The extracellular matrix consists ofa complex structure and is constituted, inter alia, of collagen,proteoglycans, glycoproteins and glycosaminoglycans. Degradation of theextracellular matrix is essential to many biological processes, such asembryo-genesis, morphogenesis and tissue absorption and remodelling.However, uncontrolled activity of the MMPs can result in a considerabledestruction of tissues, such as in rheumatic arthritis (Okada et al.,1986).

[0005] Further biological functions of TIMP-2 are related to theinhibition of the matrix metalloproteinases, including, inter alia,reduction of the growth of tumor cells (Gomez et al., 1997), andinhibition of angiogenesis (Valente et al., 1998).

[0006] In addition to these functions, intrinsic biological activitiesof TIMP-2 have also been described. These include an increased formationof red blood cells (Stetler-Stevenson et al., 1992), and a mitogeniceffect on different cell lines (Hayakawa et al., 1994). Further, bothinhibition of bone absorption in whole bone cultures (Hill et al., 1993)and stimulation of bone absorption by osteoclasts (Shibutani et al.,1999) could be shown in vitro.

[0007] FIGS. 1: A) to D) The different chromatographic steps for thepurification of osteoproliferative TIMP-2.

[0008]FIG. 2: A) Proliferative effect of recombinant human TIMP-2 onprimary fetal rat osteoblasts. B) Influence of recombinant human TIMP-2on the average cell diameter in the mouse osteoblastic cell lineMC3T3-E1.

[0009] Surprisingly, TIMP-2 having the amino acid sequenceZ¹CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGPX1KDTEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTLX2TTQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEKX3INGHQAKFFACIKR SDGSCAWYRGAAPPKQEFLDIEDP-Z²

[0010] wherein X1 represents the amino acid E or D; X2 represents theamino acid T or I; X3 represents the amino acid N or S; Z¹ represents—NH₂, a substituted amine or an arbitrary peptide having up to ten aminoacids; Z² represents —COOH, CONH₂, a substituted amide or an arbitrarypeptide having up to ten amino acids;

[0011] and its biologically active fragments and/or amidated, acylated,sulfated, phosphorylated, glycosylated and/or polyethylene-glycolmodified derivatives, are capable of causing stimulation of theosteoblasts responsible for osteogenesis. TIMP-2 has a proliferative andprotective effect on fetal osteoblasts.

[0012] Therefore, according to the invention, a medicament is claimedcontaining TIMP-2 having the amino acid sequenceZ¹CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGPX1KDIEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTLX2TTQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEKX3INGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIED P-Z²

[0013] wherein X1 represents the amino acid E or D; X2 represents theamino acid T or I; X3 represents the amino acid N or S; Z¹ represents—NH₂, a substituted amine or an arbitrary peptide having up to ten aminoacids; Z² represents —COOH, CONH₂, a substituted amide or an arbitrarypeptide having up to ten amino acids;

[0014] and its biologically active fragments and/or amidated, acylated,sulfated, phosphorylated, glycosylated and/or polyethylene-glycolmodified derivatives.

[0015] According to the invention, there are preferably employed humanTMP-2 (SEQ ID NO. 1) and rat TIMP-2 (SEQ ID NO. 2):CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGPEKD (SEQ. ID.NO 1) IEFIYTAPSSAVCGVLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTLSTTQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEKNINGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDPCSCSPVHPQQAFCNADVVIPAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGPDKD (SEQ. ID. NO2) IEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTLSITQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEKSINGHQAKFFACIKRSDGSCAWYRGAAPPKQEFLDIEDP

[0016] The invention also relates to the use of TIMP-2 having the aminoacid sequence Z¹CSCSRVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGPX1KDIEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTLX2TTQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEKX3INGHQAKFFACIKRSDGSCAWYRGAAPP KQEFLDIED P-Z²

[0017] wherein X1 represents the amino acid E or D; X2 represents theamino acid T or I; X3 represents the amino acid N or S; Z¹ represents—NH₂, a substituted amine or an arbitrary peptide having up to ten aminoacids; Z² represents —COOH, CONH₂, a substituted amide or an arbitrarypeptide having up to ten amino acids;

[0018] and its biologically active fragments and/or amidated, acylated,sulfated, phosphorylated, glycosylated and/or polyethylene-glycolmodified derivatives for preparing an osteoanabolic medicament.

[0019] TIMP-2 derivatives which can be used according to the inventionpreferably have at least 90% sequence identity with the native sequenceof TIMP-2 or the mentioned modifications.

[0020] It may be advantageous to employ the TIMP-2 to be used accordingto the invention in combination with other growth factors and/ormedicaments and in combination with medical aids, for example, ingrafts.

[0021] The administration of TIMP-2 is effected, in particular, as aformulation in the form of injections, ointments, sustained releasecapsules and similar galenic formulations.

[0022] The invention also relates to the use of low-molecular weightsubstances and active compounds which have effects on bone cellscomparable to those of TIMP-2 for preparing a medicament for treatingdiseases of the bone system, such as fractures, osteopenia andosteoporosis.

[0023] Further, the invention also relates to the use of a receptor forTIMP-2 on bone cells in connection with its related agonists andantagonists for preparing a medicament for diseases of the bone system,such as fractures, osteopenia and osteoporosis.

[0024] The TIMP-2 to be used according to the invention is employed, inparticular, for preparing an osteoanabolic medicament for treating bonedefects and for improving bone regeneration, especially following bonefractures or surgical intervention, and for treating degenerative andmetabolic bone diseases, such as osteoporosis, osteopenia andosteomalacia, and inflammatory bone diseases, such as ostitis andosteomyelitis.

[0025] The invention also relates to a diagnostic agent for functionaldisorders of the bone, containing TIMP-2 having the amino acid sequenceZ¹CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGPX1KDIEFIYTAPSSAVCGSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTLX2TTQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEKX3INGHQAKFFACIKRSDGSCAWYRGAAPP KQEFLDIEDP-Z²

[0026] wherein X1 represents the amino acid E or D; X2 represents theamino acid T or I; X3 represents the amino acid N or S; Z¹ represents—NH₂, a substituted amine or an arbitrary peptide having up to ten aminoacids; Z² represents —COOH, CONH₂, a substituted amide or an arbitrarypeptide having up to ten amino acids;

[0027] and its biologically active fragments and/or amidated, acylated,sulfated, phosphorylated, glycosylated and/or polyethylene-glycolmodified derivatives.

[0028] The basis of the identification of TIMP-2 as an osteoanabolicpeptide was the observation that there is a different ability ofregeneration of bone defects depending on the age of an organism. Whileadult organisms are not normally capable of regenerating major bonedefects by the formation of new bone tissue, a complete bonyregeneration of comparable defects takes place in younger organisms.

[0029] Similar observations were also made in vitro in the examinationof osteoblasts: Osteoblasts isolated from fetal organisms grow withoutany problems in cultures and begin to mineralize after a few days. Incontrast, osteoblasts from immature and adult animals are not capable ofproliferating under standard culture conditions and eventually die. Ifthese cells, which are not viable alone, are supplemented with thesupernatant of fetal osteoblasts, they proliferate and mineralize in away similar to the fetal cells.

[0030] A clearly regenerative capability of the cell culture supernatantof fetal osteoblasts could also be shown in vivo. Thus, a skull defectwhich cannot heal with the formation of bone tissue without externalstimulation was surgically caused to rats. However, if a peptide extractfrom the cell culture supernatant of fetal primary osteoblasts isadministered into these defects, then a complete regeneration of thedefect takes place with the formation of new bone tissue.

[0031] In order to isolate the substance which is responsible for theeffects described above, a peptide extraction from the supernatant offetal osteoblasts was performed, and the peptides obtained weresubsequently examined in vitro for their osteoproliferative capabilities(see Example 3). The peptide according to the invention could bepurified thereby using preparative, semipreparative and analytical RPchromatography due to its biological activity.

[0032] The biochemical characterization of the purified peptideaccording to the invention was effected by mass spectrometry andN-terminal sequencing. Determination of the molecular mass byelectrospray mass spectrometry yielded a molecular mass of 21 715 Da.The N-terminal sequence analysis by Edman degradation resulted in thefollowing sequence:

[0033] XSXSPVHPQQAFXNADVVIRAKAV

[0034] The substance according to the invention can be employed toinduce enhanced growth and improved regeneration of bone tissue. Thismay be considered, in particular, after bone fractures or surgicalintervention, but also in systemic bone diseases.

[0035] The invention will be illustrated in more detail by the followingExamples:

EXAMPLE 1 Isolation of TIMP-2 from Cell Culture Supernatant

[0036] Cell culture supernatants were obtained from confluent culturesof primary fetal osteoblasts. Thus, the osteoblasts were first isolatedfrom the skull caps of rat fetuses by sequential digestion withcollagenase and trypsin, followed by cultivation in Eagle's minimalessential medium (MEM) with penicillin/streptomycin and 10% fetal calfserum (FCS). After confluency was reached, the cells were keptalternately with 10% FCS for 24 hours and serum-free for 24 hours. Theserum-free supernatants were collected and used for further peptideisolation.

[0037] The isolation of TIMP-2 was effected by various subsequentRP-HPLC steps which served for the fractioning of the whole peptideextract. Aliquots of the fractions were tested in a bioassay. Thefractions were stored at −20° C.

[0038] The different chromatographic steps for the purification ofosteoproliferative TIMP-2 are shown in FIGS. 1 A-D.

[0039] 1. Preparative Chromatography

[0040] The serum-free cell culture supernatants were adjusted with HCIto a pH of 2.5 and by the addition of acetonitrile to 5% acetonitrile.Subsequently, they were filtered first over a glass fiber filter (GF6,Schleicher & Schuell) and then over a membrane filter (OE G7, 4.43 μm,Schleicher & Schuell). The material thus obtained was charged onto a RPcolumn in amounts of from 1 to 21 with a flow rate of from 40 to 50ml/min.

[0041] Chromatographic conditions: Column: YMC Gel Basic (15-30 μm, 47 ×300 mm) Flow: 40 ml/min Fractions: 50 ml Buffer A: 10 mM HCl Buffer B:80% (w/v) acetonitrile, 10 mM HCl Gradient: 5 to 75% B in 47.5 min 75 to100% B in 5 min.

[0042] Subsequently, aliquots of the fractions obtained were tested in abioassay.

[0043] 2. Semipreparative Separation

[0044] Fractions 37-40, which were bioactive in the assay, wereseparated by means of a further RP step.

[0045] Chromatographic conditions: Column: Biotek RP Silica C4 (100 A, 5μm, 20 × 125 mm) Flow: 5 ml/min Fraction: 5 ml Buffer A: 0.10% TFABuffer B: 80% (w/v) acetonitrile, 0.1% TFA Gradient: 5 to 40% B in 10min 40 to 100% B in 60 min.

[0046] Aliquots of the fractions obtained were again tested in abioassay.

[0047] 3. Analytical Chromatography

[0048] The subsequent fractioning of the bioactive fractions 22 and 23was effected by means of another RP chromatography.

[0049] Chromatographic conditions: Column: YMC C18 (120 A, 5 μm, 4.6 ×250 mm) Flow: 0.6 ml/min Fraction: 0.6 ml Buffer A: 0.1% TFA Buffer B:80% (w/v) acetonitrile, 0.1% TFA Gradient: 5 to 30% B in 4 min 30 to 55%B in 50 min 55 to 100% B in 5 min.

[0050] Aliquots of the fractions obtained were again tested in abioassay.

[0051] 4. Analytical Chromatography

[0052] The bioactive fractions 27-29 were again separated by means of RPchromatography.

[0053] Chromatographic conditions: Column: Phenomenex C5 (4.6 × 250 mm)Flow: 0.6 ml/min Fraction: 0.6 ml Buffer A: 0.1% TFA Buffer B: 80% (w/v)acetonitrile, 0.1% TFA Gradient: 5 to 38% B in 4 min 38 to 58% B in 60min 58 to 100% B in 5 min.

[0054] In the subsequent biotest, the peptide according to the inventioncould be obtained in pure form in fraction 24.

EXAMPLE 2 Biochemical Analysis of TIMP-2

[0055] 1. Mass Determination

[0056] The mass determination of the native purified peptide wasperformed by means of an ESI mass spectrometer. A molecular mass of 27715 Da was obtained. This is in very good agreement with the theoreticalmass of the oxidized form of TIMP-2 in which all the cysteines arebridged by cystine bridges.

[0057] 2. Sequence Determination

[0058] The purified native peptide was analyzed by Edman degradation onan ABI 494 sequencer using the standard program. The first 24degradation steps yielded the following N-terminal sequence:

[0059] XSXSPVHPQQAFXNADVVIRAKAV

[0060] X are cysteine residues which cannot be detected by sequencing.Taking into account the non-detectable cysteines, the sequence obtainedis exactly identical with the known sequence of TIMP-2. Minor sequenceswere not obtained.

[0061] 3. Data Base Search

[0062] In a subsequent alignment in the data base SwissProt, thesequence was unambiguously identified as the beginning of TIMP-2. Thetheoretical mass of the protein of 27 713 Da is also in agreement withthe measured mass of 27 715 Da. Thus, the purified peptide could beunambiguously identified as TIMP-2.

EXAMPLE 3 Determination of the Biological Peptide of TIMP-2

[0063] The isolation of TIMP-2 was effected by means of its biologicalactivity, the proliferation of fetal rat osteoblasts being measured.

[0064] Thus, aliquots of the peptide fractions obtained were lyophilizedand resuspended in serum-free medium. Subsequently, their proliferativeeffect on fetal osteoblasts was tested. Subsequently, fractionsexhibiting a proliferative effect were subjected to anotherchromatographic separation. The different chromatographic steps for thepurification of osteoproliferative TIMP-2 are shown in FIGS. 1 A-D.

[0065] For proliferation measurement, the primary osteoblasts weretrypsinized and plated in 96 well plates in a serum-free medium in acell density of 5000 cells per well. Subsequently, the fractions to betested were added. After an incubation time of 48 h or 72 h, theproliferation was tested by means of two different methods. As positivecontrols, different concentrations of fetal calf serum and TGFβ1 wereused. Cells having obtained no stimulation served as a negative control.

[0066] On the one hand, proliferation measurement was effected by meansof the Wst-1 substrate. This substrate is converted by mitochondrialenzymes in metabolically active cells to a colored product, the colorintensity formed being proportional to the number of cells. The colorintensity is determined at a wavelength of 405 nm and a referencewavelength of above 600 nm in an ELISA reader.

[0067] On the other hand, the proliferation is measured by directmeasurement of the cell number in a Coulter counter (CASY).

[0068] Both methods showed a dose-dependent growth-promoting effect ofrecombinant human TIMP-2 on osteoblasts (FIG. 2A). A significantosteoproliferative effect of TIMP-2 can be detected in these in-vitroexperiments from concentrations of 100 ng/ml. In addition, human TIMP-2causes a decrease of the average cell diameter in the mouse osteoblasticcell line MC3T3-E1 (FIG. 2B).

[0069] Significant effects of TIMP-2 can be detected in this in-vitroexperiment from concentrations of 100 ng/ml. Further, a protective andproliferative effect on the mouse osteoblastic cell line MC3T3-E1 couldbe shown by microscopy. Since all these cells are typical bone cells,TIMP-2 can be considered an osteoanabolic factor.

1 4 1 194 PRT Homo sapiens 1 Cys Ser Cys Ser Pro Val His Pro Gln Gln AlaPhe Cys Asn Ala Asp 1 5 10 15 Val Val Ile Arg Ala Lys Ala Val Ser GluLys Glu Val Asp Ser Gly 20 25 30 Asn Asp Ile Tyr Gly Asn Pro Ile Lys ArgIle Gln Tyr Glu Ile Lys 35 40 45 Gln Ile Lys Met Phe Lys Gly Pro Glu LysAsp Ile Glu Phe Ile Tyr 50 55 60 Thr Ala Pro Ser Ser Ala Val Cys Gly ValSer Leu Asp Val Gly Gly 65 70 75 80 Lys Lys Glu Tyr Leu Ile Ala Gly LysAla Glu Gly Asp Gly Lys Met 85 90 95 His Ile Thr Leu Cys Asp Phe Ile ValPro Trp Asp Thr Leu Ser Thr 100 105 110 Thr Gln Lys Lys Ser Leu Asn HisArg Tyr Gln Met Gly Cys Glu Cys 115 120 125 Lys Ile Thr Arg Cys Pro MetIle Pro Cys Tyr Ile Ser Ser Pro Asp 130 135 140 Glu Cys Leu Trp Met AspTrp Val Thr Glu Lys Asn Ile Asn Gly His 145 150 155 160 Gln Ala Lys PhePhe Ala Cys Ile Lys Arg Ser Asp Gly Ser Cys Ala 165 170 175 Trp Tyr ArgGly Ala Ala Pro Pro Lys Gln Glu Phe Leu Asp Ile Glu 180 185 190 Asp Pro2 162 PRT Homo sapiens 2 Cys Ser Cys Ser Pro Val His Pro Gln Gln Ala PheCys Asn Ala Asp 1 5 10 15 Asn Asp Ile Tyr Gly Asn Pro Ile Lys Arg IleGln Tyr Glu Ile Lys 20 25 30 Thr Ala Pro Ser Ser Ala Val Cys Gly Val SerLeu Asp Val Gly Gly 35 40 45 Lys Lys Glu Tyr Leu Ile Ala Gly Lys Ala GluGly Asp Gly Lys Met 50 55 60 His Ile Thr Leu Cys Asp Phe Ile Val Pro TrpAsp Thr Leu Ser Ile 65 70 75 80 Thr Gln Lys Lys Ser Leu Asn His Arg TyrGln Met Gly Cys Glu Cys 85 90 95 Lys Ile Thr Arg Cys Pro Met Ile Pro CysTyr Ile Ser Ser Pro Asp 100 105 110 Glu Cys Leu Trp Met Asp Trp Val ThrGlu Lys Ser Ile Asn Gly His 115 120 125 Gln Ala Lys Phe Phe Ala Cys IleLys Arg Ser Asp Gly Ser Cys Ala 130 135 140 Trp Tyr Arg Gly Ala Ala ProPro Lys Gln Glu Phe Leu Asp Ile Glu 145 150 155 160 Asp Pro 3 194 PRTArtificial Consesus sequence for TIMP-2 3 Cys Ser Cys Ser Pro Val HisPro Gln Gln Ala Phe Cys Asn Ala Asp 1 5 10 15 Val Val Ile Arg Ala LysAla Val Ser Glu Lys Glu Val Asp Ser Gly 20 25 30 Asn Asp Ile Tyr Gly AsnPro Ile Lys Arg Ile Gln Tyr Glu Ile Lys 35 40 45 Gln Ile Lys Met Phe LysGly Pro Xaa Lys Asp Ile Glu Phe Ile Tyr 50 55 60 Thr Ala Pro Ser Ser AlaVal Cys Gly Val Ser Leu Asp Val Gly Gly 65 70 75 80 Lys Lys Glu Tyr LeuIle Ala Gly Lys Ala Glu Gly Asp Gly Lys Met 85 90 95 His Ile Thr Leu CysAsp Phe Ile Val Pro Trp Asp Thr Leu Xaa Thr 100 105 110 Thr Gln Lys LysSer Leu Asn His Arg Tyr Gln Met Gly Cys Glu Cys 115 120 125 Lys Ile ThrArg Cys Pro Met Ile Pro Cys Tyr Ile Ser Ser Pro Asp 130 135 140 Glu CysLeu Trp Met Asp Trp Val Thr Glu Lys Xaa Ile Asn Gly His 145 150 155 160Gln Ala Lys Phe Phe Ala Cys Ile Lys Arg Ser Asp Gly Ser Cys Ala 165 170175 Trp Tyr Arg Gly Ala Ala Pro Pro Lys Gln Glu Phe Leu Asp Ile Glu 180185 190 Asp Pro 4 24 PRT Rattus rattus misc_feature (1)..(1) Xaa can beany naturally occurring amino acid 4 Xaa Ser Xaa Ser Pro Val His Pro GlnGln Ala Phe Xaa Asn Ala Asp 1 5 10 15 Val Val Ile Arg Ala Lys Ala Val 20

1. A medicament containing TIMP-2 having the amino acid sequenceZ¹CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGPX1KDIEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTLX2TTQKKSLNHRYQNGCECKITRCPMIPCYISSRDECLWMDWVTEKX3INGHQAKFFACIKRSDGSCAWYRGAAPP KQEFLDIED P-Z²

wherein X1 represents the amino acid E or D; X2 represents the aminoacid T or I; X3 represents the amino acid N or S; Z¹ represents —NH₂, asubstituted amine or an arbitrary peptide having up to ten amino acids;Z² represents —COOH, CONH₂, a substituted amide or an arbitrary peptidehaving up to ten amino acids;

and its biologically active fragments and/or amidated, acylated,sulfated, phosphorylated, glycosylated and/or polyethylene-glycolmodified derivatives.
 2. Use of TIMP-2 having the amino acid sequenceZ¹CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGPX1KDIEFIYTAPSSAVCGVSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTLX2TTQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEKX3TNGHQAKFFACIKRSDGSCAWYRGAAPP KQEFLDIEDP-Z²

wherein X1 represents the amino acid E or D; X2 represents the aminoacid T or I; X3 represents the amino acid N or S; Z¹ represents —NH₂, asubstituted amine or an arbitrary peptide having up to ten amino acids;Z² represents —COOH, CONH₂, a substituted amide or an arbitrary peptidehaving up to ten amino acids;

and its biologically active fragments and/or amidated, acylated,sulfated, phosphorylated, glycosylated and/or polyethylene-glycolmodified derivatives for preparing an osteoanabolic medicament.
 3. Theuse according to claim 1, wherein said TIMP-2 derivative has at least90% sequence identity with the sequence according to claim
 1. 4. The useof TIMP-2 according to claims 1 to 3 in combination with other growthfactors and/or medicaments and in combination with medical aids, forexample, in grafts.
 5. The use of TIMP-2 according to claims 1 to 4formulated for injections, ointments, sustained release capsules andsimilar galenic formulations.
 6. Use of low-molecular weight substancesand active compounds which have effects on bone cells comparable tothose of TIMP-2 for preparing a medicament for treating diseases of thebone system, such as fractures, osteopenia and osteoporosis.
 7. Use of areceptor for TIMP-2 on bone cells in connection with its relatedagonists and antagonists for preparing a medicament for treatingdiseases of the bone system, such as fractures, osteopenia andosteoporosis.
 8. The use according to claims 1 to 7 for preparing anosteoanabolic medicament for treating bone defects and for improvingbone regeneration, especially following bone fractures or surgicalintervention, and for treating degenerative and metabolic bone diseases,such as osteoporosis, osteopenia and osteomalacia, and inflammatory bonediseases, such as ostitis and osteomyelitis.
 9. A diagnostic agent forfunctional disorders of the bone, containing TIMP-2 having the aminoacid sequence Z¹CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQIKMFKGPx1KDIEFIYTAPSSAVCGSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTLX2TTQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEKX3INGHQAKFFACIKRSDGSCAWYRGAAPP KQEFLDIED P-Z²

wherein X1 represents the amino acid E or D; X2 represents the aminoacid T or I; X3 represents the amino acid N or S; Z¹ represents —NH₂, asubstituted amine or an arbitrary peptide having up to ten amino acids;Z² represents —COOH, CONH₂, a substituted amide or an arbitrary peptidehaving up to ten amino acids;

and its biologically active fragments and/or amidated, acylated,sulfated, phosphorylated, glycosylated and/or polyethylene-glycolmodified derivatives.
 10. Use of TIMP-2 having the amino acid sequenceZ¹CSCSPVHPQQAFCNADVVIRAKAVSEKEVDSGNDIYGNPIKRIQYEIKQTKMFKGPX1KDIEFIYTAPSSAVCGSLDVGGKKEYLIAGKAEGDGKMHITLCDFIVPWDTLX2TTQKKSLNHRYQMGCECKITRCPMIPCYISSPDECLWMDWVTEKX3INGHQAKFFACIKRSDGSCAWYRGAAPP KQEFLDIED P-Z²

wherein X1 represents the amino acid E or D; X2 represents the aminoacid T or I; X3 represents the amino acid N or S; Z¹ represents —NH₂, asubstituted amine or an arbitrary peptide having up to ten amino acids;Z² represents —COOH, CONH₂, a substituted amide or an arbitrary peptidehaving up to ten amino acids;

and its biologically active fragments and/or amidated, acylated,sulfated, phosphorylated, glycosylated and/or polyethylene-glycolmodified derivatives .as a diagnostic agent in functional disorders ofthe bone.